高灵敏度电化学发光PCR方法检测基因点突变研究(英文)

Study on the Highly Sensitive ECL-PCR Method for Point Mutation Detection

近年来发展了一种用于定量检测基因点突变的电化学发光PCR方法。该法采用三联吡啶钌标记的上游引物和生物素标记的下游引物对待测基因进行PCR扩增;随后,采用特定的限制性内切酶对扩增产物进行酶切,由于野生型样品和突变型样品间存在酶切位点的变化,其中只有一种基因型样品能被切断;通过生物素与链霉亲和素包被的磁珠连接,将生物素标记的DNA片段收集到样品池中;进行电化学发光检测,通过所得信号的有无可以判断其基因型。我们分别将该法用于Presenilin-1基因和H-ras癌基因的点突变检测,结果均可明显区分突变型样品和野生型样品。该法具有灵敏、快速、简便、安全等优点,是一种实用的基因点突变检测方法。

Recently, we have developed an electrochemiluminescence-polymerase chain reaction （ECL-PCR） method for point mutation detection. In this method, the target gene was amplified by a Ru（ bpy）3^2＋ （TBR）-labeled forward and a biotinylated reverse primer, and then followed by digestion with a kind of restriction enzyme, which only cut the wildtype （or mutant） amplicon containing its recognition site. Reaction product was detected by electrochemiluminescence （ECL） assay after adsorption of the resulting DNA duplex to the solid phase. One strand of PCR products carries biotin to be bound on a streptavidin-coated microbead for sample selection. Another strand carries TBR to react with tripropylamine （TPA） and emit light for ECL detection. The method was applied to detect the point mutation in presenilin-1 and H-ras genes. The two genotypes were clearly discriminated. In summary, the ECL-PCR method can be used to detect a point mutation that creates or destroys a restriction site in any gene. It is useful in point mutation detection due to its sensitivity, rapidness, simplicity and safety.